Pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus is necessary for proper reproductive function. Despite its importance, very little information is available on the cellular basis for this phenomenon because of the difficulty in studying the few and scattered GnRH neurons present in hypothalamic tissue. Recent development of GT1 clonal neuronal cultures that release GnRH in a pulsatile manner provided an excellent model system to study this process. Our efforts during the last grant period were directed toward developing a series of tools that could be used in conjunction with these cells to begin to elucidate the cellular components that contributed to pulsatile GnRH release. In this regard, we developed tools using a promoter-driven luciferase reporter for measurement of gene expression, fura-2 for identification of intracellular calcium changes, and FM1-43 for assessment of exocytotic function. These tools were unique in that (whether applied singly or in sequence) each was designed to measure activity only individual cells and in real-time. Using this approach, we found that episodic activity in GT1-7 cells was not just restricted to secretion, but that calcium changes and gene expression also occurred in pulses. More importantly though, our findings indicated that GnRH episodic activity was governed by two cell processes; an intrinsic pulsatile function of individual neurons and a coordination of multiple neurons to achieve distinct collective pulses of release. These findings provided the first experimental evidence of a process by which hypothalamic neurons may achieve the critical role of episodic GnRH release. We believe that the most effective means to study this role is by focusing individually on each of these components. Thus, our specific aims in this proposal are 1) to determine what cellular and molecular mechanisms underlie intermittent activity in individual GnRH neurons and 2) to elucidate the processes underlying GnRH neuronal cell synchronization. Experiments directed toward achieving these aims should provide important insight in to the principles governing the cellular basis for GnRH pulsatility. These principles may be invaluable in devising future therapeutic strategies that can be used to correct reproductive disfunction resulting from abnormal GnRH release. [unreadable] [unreadable]